Microarray and Proteomic Studies:[unreadable] We have investigated the protein expression profiles in Fabry cells using microarray and proteomics technology. Gene expression analyses of the heart, aorta, and liver of male alpha-galactosidase A knockout mice before and after multiple injections of alpha-galactosidase A demonstrated an alteration of Rpgrip1. Enzyme replacement therapy tended to normalize gene expression. Future developments in array technology for proteins and DNA single nucleotide polymorphism analysis, together with gene expression microarray analysis, will open a new chapter in our understanding of the biology of lysosomal storage disorders. [unreadable] [unreadable] Cultured Endothelial Cell Model:[unreadable] A cultured endothelial cell model with an extended lifespan was created as a critical element for future study of the vasculopathy of Fabry disease. The cell line has markedly extended lifespan compared to parental primary cell, continues to stably express many key markers of endothelial cells and retains many functional characteristics. The cells show significantly reduced activity of alpha-galactosidase A compared with primary endothelial cells from normal individuals and accumulate globotriaosylceramide in lysosomes. This cell line will provide a useful in vitro model of Fabry disease and will facilitate systematic studies to investigate pathogenic mechanisms and explore new therapeutic approaches for Fabry disease.[unreadable] [unreadable] Viral Based Vectors for Gene Correction:[unreadable] We first demonstrate LV-mediated marking of peripheral blood (PB) cells by transduction/transplantation of hematopoietic stem/progenitor cells. Fabry mice were transplanted with bone marrow mononuclear cells (BMMNCs) transduced a single time with an LV encoding the human alpha-gal A cDNA. Sustained expression of functional alpha-gal A in Fabry mice was observed over 24 weeks and reduction of globotriaosylceramide was observed in all organs assessed. [unreadable] [unreadable] Enzyme Replacement Therapy (ERT):[unreadable] Distribution of alpha-galactosidase A after intravenous infusion in mice, investigated using immunohistochemically, demonstrated specific staining in liver, kidney, heart, testes, adrenal gland, spleen and bone marrow. There was no difference in distribution of the infused enzyme distribution among tissues sampled 4, 24, and 48h post-injection. The intracellular localization of immunopositivity varied considerably between organs with vascular endothelium being the most commonly positive site. We conclude that intravenously injected alpha-Galactosidase A has a very heterogeneous systemic distribution. [unreadable] Enzyme Replacement Therapy: In collaboration with members of the Surgical Neurology Branch (SNB), we determined the safety of intracerebral injection of GC in non-human primates using the convection-enhanced delivery (CED) technique developed by SNB. [unreadable] To determine if CED of glucocerebrosidase could be used to treat targeted sites of disease progression in the brain and brainstem of a patient with neuronopathic Gaucher disease while monitoring enzyme distribution using MRI. The safety and feasibility of this delivery and monitoring paradigm were evaluated. Animal studies revealed that real-time, T1-weighted, MRI of Gd-DTPA accurately tracked enzyme distribution during CED. Targeted perfusion of clinically affected anatomic sites in a patient with neuronopathic Gaucher disease (frontal lobe and brainstem) with glucocerebrosidase was successfully performed. The patient tolerated the infusions without evidence of toxicity. Thus, convection-enhanced delivery can be used to safely perfuse large regions of the brain and brainstem with therapeutic levels of glucocerebrosidase. Co-infused imaging surrogate tracers can be used to monitor and control the distribution of therapeutic agents in vivo. Patients with neuronopathic Gaucher disease and other intrinsic CNS disorders may benefit from a similar treatment paradigm.[unreadable] [unreadable] Pharmacological Chaperone Therapy: [unreadable] We developed a rapid screening assay for enhancement of endogenous alpha-galactosidase A (alpha-Gal A) in patient-derived cells. We used a T-cell based system to screen 40 mutations causing Fabry disease for enhanceability using 1-deoxygalactonojirimycin (DGJ). 17 mutations were found to be enhanceable to at least 25% of normal using this test system. Mutations located within alpha-helical or beta-sheets in the moleculenwere less likely to be enhanceable compared with mutations located in intervening sequences outside these regions. The level of activity achieved provides a basis for the therapeutic trial of DGJ in patients with similarly enhanceable enzyme. This assay method has general utility in other disorders in assessing the degree of enhancement of activity of mutated proteins by PCT and the identification of the pattern of enhanceability provides a rational basis for increased screening of patients who are more likely to benefit.[unreadable] [unreadable] [unreadable] Protein Transduction Domains: [unreadable] We are extending our strategies to extend the organ and tissue distribution of exogenous enzymes for the treatment of both Gaucher disease and Fabry disease to augment the effectiveness of enzyme replacement therapy. We have previously expressed a TAT-GC fusion protein in which recombinant glucocerebrosidase (GC) is fused to 11-amino acid peptide from the HIV-1 transactivator protein (TAT) which functions to facilitate the transport of the enzyme across the plasma membrane of a variety of cell-types in a receptor-independent manner. We have extended these studies to include additional GC constructs containing flexible spacers to increase enzyme activity in addition to a variety of TAT analogs in order to test the effect of sequence variation on the uptake of the enzyme. Further studies have also been undertaken to construct similar fusions with the enzyme alpha-galactosidase A (AGA) as a possible strategy for wider distribution of this enzyme for the treatment of Fabry disease. Efforts are underway to introduce these fusion proteins into animals to test their distribution and compare these findings with that of the native constructs. Recombinant GC-TAT and AGA-TAT were expressed in eukaryotic cells from which catalytically active, normally glycosylated enzyme fusion proteins were obtained and tested for receptor independent uptake into cultured cells. It is expected that GC-TAT will be more efficiently delivered than unmodified GC to cells in the bone marrow and lung, and perhaps additional cells that lack the mannose lectin and thereby enhance the clinical responses of patients with Gaucher to enzyme replacement therapy. The AGA-TAT enzyme may be capable of entering heart and kidney more efficiently than the native enzyme. Alterations in the enzyme aimed at improving the stability of the administered enzyme are currently under investigation. [unreadable] Glucosyl Transferase and the Pathogenesis of Gaucher Disease: GlcSph is neurotoxic and contributes significantly to the dysfunction and destruction of brain cells. Our investigations have identified six compounds that inhibit the enzymatic synthesis of GlcSph and ranked them in the order of their effectiveness. Using compounds that reach the brain in an effective and non-toxic concentration, we propose to conduct clinical trials with these agents to improve the debilitating clinical course in patients with neuronopathic Gaucher disease.